Pharmaceutical formulations comprising substituted xanthine compounds

ABSTRACT

The present invention provides novel pharmaceutical compositions comprising substituted xanthine compounds useful for the treatment of cystic fibrosis and other diseases, and methods of use thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel pharmaceutical formulations.Specifically, the present invention provides novel formulations ofsubstituted xanthine compounds for the treatment of cystic fibrosis, andother diseases, including chronic obstructive pulmonary diseases(COPDs).

2. Description of the Related Art

Cystic Fibrosis and COPD

Cystic fibrosis (CF) is the most common fatal genetic disease affectingthe Caucasian population. The incidence of the disease among CaucasianAmericans is approximately 1 of every 2500 live births. AmongAfro-Americans, the incidence is less frequent, with about 1 of every17,000 live births. An estimated 70,000 victims suffer from the diseaseworldwide. Apart from the loss of life and loss of quality of life, itcosts about $50,000 a year to treat a cystic fibrosis patient in theUnited States, mostly using antibiotics, enzyme, and other drugs thathelp prolong life, but inevitably fail to save it.

Cystic fibrosis is a whole body disease, and the associatedabnormalities are many and varied, due to the multi-systemic nature ofthe disease. Most of the diverse symptoms displayed are attributed tounderlying abnormality in exocrine gland function. Three general typesof pathophysiology are observed in the exocrine glands of cysticfibrosis patients. These are (1) glands become obstructed due to viscidor solid material in the luminal space of the gland (e.g., as observedin the pancreas and intestinal glands), (2) glands are histologicallyabnormal and produce an excess of secretions (e.g., tracheobronchialglands), and (3) glands are histologically normal, but secrete excessivesodium (Na⁺) and chloride (CF) ions (e.g., the sweat glands).

Signs of the disease can manifest from the time of birth, and can varywidely in their severity. Inevitably, all patients suffering from thedisease develop chronic progressive disease of the respiratory system,characterized by accumulation of excessively viscous mucus secretion,airway plugging, and opportunistic bacterial infection in the airway.Although many organ systems are affected, approximately 90% of patientseventually succumb to pulmonary failure exacerbated by chronicinfection. In the majority of cases, pancreatic dysfunction occurs, andhepatobiliary and genitourinary diseases, including infertility, arealso manifested. Although survival of cystic fibrosis patients hasimproved in recent years, the median survival is still only about 30years despite the development and implementation of intensive supportiveand prophylactic treatment

The pulmonary complications of cystic fibrosis are one example of alarger category of diseases, namely, those diseases that result inchronic obstruction of the airway, which includes the alveoli, bronchi,bronchioles and upper airway, including the trachea. Collectively, thesedisorders are broadly termed chronic obstructive pulmonary disease(COPD), or synonymously, chronic obstructive airway disorders (COAD),regardless of disease etiology. COPD encompasses various diseases, allof which share the common pathology of airway obstruction. The diseasesthat can manifest as COPD's can include, for example, cystic fibrosis,chronic bronchitis, emphysema and asthma. Furthermore, patientsdisplaying COPD pathology may have complex and overlapping etiologies,for example, in asthmatic bronchitis. Treatment for COPD often usesbronchodilator drugs, which may offer some relief to the patient,regardless of disease etiology. Anti-inflammatory agents, antibioticsand/or oxygen therapy are also appropriate for some COPD patients.

The CFTR Gene and Gene-Product

Cystic fibrosis disease is caused by mutations in the cystic fibrosistransmembrane regulator (CFTR) gene. The most common of these mutations,accounting for approximately 75% of mutant CFTR alleles, results in thedeletion of a phenylalanine residue at position 508 (written ΔPhe⁵⁰⁸ orΔF508). More than 900 different mutations have been identified in theremaining 25% of the mutant CFTR alleles (Kunzelmann and Nitschke, Exp.Nephrol., 8:332-342 [2000]).

The ΔF508 mutation commonly found in CFTR alleles is located within thefirst nucleotide binding fold (NBF-1) of the CFTR protein (Schoumacheret al., Proc. Natl. Acad. Sci., 87:4012-4016 [1990]; Riordan et al.,Science 245:1066-1073 [1979]). More specifically, the ΔF508 mutation islocated in a portion of the NBF-1, flanked on the N-terminal side byamino acid position 458-471 (known as the Walker A sequence) and on theC-terminal side by amino acid position 548-560 (known as the C-domain),and further by amino acid position 561-573 (known as the Walker Bdomain). The physiological function of the CFTR amino acids locatedbetween positions 471 and 561 is unknown.

The regulated movement of inorganic ions across the cell membrane isrequired to maintain a proper electrical potential across cellularmembranes, as well as maintaining an appropriate intracellular ionicstrength. For example, sodium (Na⁺), chloride (Cl⁻) ions, potassium(K⁺), and calcium (Ca²⁺) ions cross animal cell membranes in such amanner that K+ and Ca²⁺ are generally accumulated intracellularly,whereas Na⁺, in large measure, is excluded from the cell interior. Themovement of these ions across the cell membrane is mediated bymembrane-bound Na⁺/K⁺ and Ca²⁺-dependent ATPases. Conductance ofchloride ions across the cell membrane is also actively regulated by atleast one ion-specific chloride channel (Edwards, Neuroscience7:1335-1366 [1982]), resulting in an under representation ofintracellular Cl⁻ relative to the overall negative intracellular charge.

The wild-type 1480 amino acid CFTR protein appears to be part of amembrane-associated cAMP-regulated chloride transporter (i.e., achloride channel) that actively secretes chloride (Cl⁻) ions acrossepithelial cell apical membranes from the cell interior to the cellexterior. Certain mutant forms of the CFTR protein, includingCFTR-ΔF508, are defective in this process. Lack of function of thenormal CFTR protein results in an abnormal charge potential across theapical surfaces of epithelial cell membranes due to reduced cellularchloride conductance. Thus, chloride, and consequently sodium, transportacross epithelial membranes of an individual expressing a mutantCFTR-ΔF508 protein is abnormal. It is also known that cells expressingthe mutant CFTR-ΔF508 protein demonstrate a higher than normalpercentage of the protein bound to the endoplasmic reticulum compared tocells expressing wild-type CFTR protein, indicating an abrogation ofCFTR trafficking, retention and degradation (Roomans, Exp. Opin. InvestDrugs 10(1):1-19 [2001]; Kunzelmann and Nitschke, Exp. Nephrol.,8:332-342 [2000]). This mutation and resulting ion conductanceimpairment as seen in cystic fibrosis patients is thought to be thecause of the cellular pathology observed in these patients, includingthe respiratory pathophysiology.

Use of Xanthine Compounds in the Treatment of Cystic Fibrosis and COPD

Various nucleotides, nucleotide derivatives, purine compounds, and mostparticularly, xanthine derivatives, show promise in stimulating chloridetransport activity, and thus, are candidate therapeutic agents in thetreatment of cystic fibrosis (Roomans, Exp. Opin. Invest. Drugs10(1):1-19 [2001]; Rodgers and Knox, Eur. Respir. J., 17:1314-1321[2001]). These xanthine compounds have a variety of advantageousactivities, including acting as pulmonary vasodilators, bronchodilatorsand smooth muscle relaxants. In addition, some of these compounds alsohave other actions, including coronary vasodilator, diuretic, cardiacand cerebral stimulant and skeletal muscle stimulant (see, U.S. Pat. No.5,032,593).

U.S. Pat. No. 4,548,818 describes the use of 3-alkyl-xanthines, such as3-cyclopentyl-3,7-dihydro-1H-purine-2,6-dione, to treat chronicobstructive airway disease (COPD), as well as cardiac disease.Di-substituted forms of xanthine are disclosed as bronchodilatoryagents. U.S. Pat. No. 5,032,593 describes the use of 1,3-alkylsubstituted 8-phenyl-xanthine compounds, such as 1-n-propyl-3-methyl-and 1-methyl-3-n-propyl-substituted xanthine derivatives, in thetreatment of bronchoconstriction.

U.S. Pat. No. 5,096,916 describes the use of imidazoline compounds inthe treatment of COPD, including cystic fibrosis, chronic bronchitis andemphysema, or COPD in association with asthma. The compound tolazolineis the preferred vasodilator compound, although other useful compoundsare also taught.

Historically, the substituted-xanthine compound theophylline has beenadministered to asthmatic and cystic fibrosis patients to enhance lungfunction. Other compounds resembling theophylline in basic structurehave been identified which possess advantageous activities, includingevoking chloride efflux from cystic fibrosis cells. These compoundsinclude 1,3-dipropyl-8-cyclopentylxanthine (CPX). CPX (and its relatedxanthine amino congeners) is a potent A1 adenosine receptor antagonistthat promotes chloride efflux from a human epithelial cell lineexpressing the CFTR-ΔF508 mutation (see, e.g., U.S. Pat. Nos. 5,366,977,5,877,179 and 6,083,954, and Eidelman et al., Proc. Natl. Acad. Sci.USA, 89:5562-5566 [1992]; Guay-Broder et al., Biochemistry34(28):9079-9087 [1995]; Jacobson et al., Biochemistry 34(28):9088-9094[1995]; Arispe et al., Jour. Biol. Chem., 273(10):5727-5734 [1998]).Based on research that originated at the National Institutes of Health,SciClone Pharmaceuticals, Inc., California, U.S., is currentlydeveloping CPX as a promising new protein-repair therapy for cysticfibrosis treatment.

Compounds related in structure to CPX and activating chloride ion effluxin cells having the ΔF508 mutation, are also known, and have beensuggested to have therapeutic value in the treatment of cystic fibrosisor other diseases. Such compounds include, for example,N,N-diallylcyclohexylxanthine (DAX; synonymously,1,3-diallyl-8-cyclohexylxanthine, DCHX),1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX), cyclohexylcaffeine(CHC), and xanthine amino congener. See, e.g., U.S. Pat. Nos. 5,366,977,5,877,179 and 6,083,954.

Accordingly, xanthine-derivatives are promising therapeutic agents forthe treatment of cystic fibrosis and other chronic obstructive airwaydisorders. A prerequisite of successful therapeutic application is,however, the development of stable pharmaceutical formulations,preferably for oral delivery, that provide good absorption andbioavailability, have suitable pharmacokinetic properties, and enablesafe administration of the therapeutically active compounds. The presentinvention meets this need by providing stable, oil-based suspensions oftherapeutically effective xanthine compounds. These formulations haveexcellent oral bioavailability and sufficient plasma half life forsuccessful use in human therapy.

These and other objects and advantages of the present invention, as wellas additional inventive features, will be apparent from the descriptionof the invention provided herein.

SUMMARY OF THE INVENTION

The invention relates to novel formulations of substituted xanthinecompounds, where the formulations are liquid formulations suitable fororal delivery. These formulations comprise at least one substitutedxanthine compound and a pharmaceutically acceptable oil. The inventionalso provides methods employing these novel formulations.

In one embodiment, the invention provides a liquid pharmaceuticalformulation suitable for oral administration comprising an effectiveamount of a therapeutically active xanthine derivative, or apharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable oil. In some embodiments, the xanthinederivative is hydrophobic. In other embodiments, the formulation is asolution, while in other embodiments, the formulation is a suspension.Where the formulation is a suspension, the xanthine derivative or apharmaceutically acceptable salt thereof, can be in the form ofparticles, and the particles optionally have a mean diameter less thanabout 100 microns. In some embodiments comprising a suspension, thesuspension is substantially homogenous.

In some embodiments, the oil in the formulation is a vegetable oil. Insome embodiments, the vegetable oil can be corn oil, almond oil, coconutoil, cottonseed oil, mustard seed oil, olive oil, palm oil, peanut oil,safflower oil, sesame oil, soybean oil, sunflower oil, and partially orfully hydrogenated derivatives of said oils. In one embodiment, corn oilis the vegetable oil.

In another embodiment, the invention provides a suspension suitable fororal administration comprising, as active ingredient, an effectiveamount of a substituted xanthine compound. It is not intended that theinvention be limited to the use of any particular substituted xanthinecompound or compounds. In this embodiment, the dispersed activeingredient is in the form of particles having a mean diameter less thanabout 100 microns.

In a related embodiment, the invention provides a suspension suitablefor oral administration comprising, as active ingredient, an effectiveamount of a substituted xanthine compound. In this embodiment, thesubstituted xanthine has the formula

(I), wherein

-   -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen, or a therapeutically        active derivative thereof, or a pharmaceutically acceptable salt        of said substituted xanthine, or a pharmaceutically acceptable        salt of said therapeutically active derivative. Furthermore in        this embodiment, the active ingredient is in the form of        particles having a mean diameter less than about 100 microns,        and the particles are dispersed in a pharmaceutically acceptable        oil. In some embodiments, the substituted xanthine is        hydrophobic. In other embodiments, the suspension is        substantially homogenous.

In various embodiments of the suspension formulation, alternatively atleast 70%, or at least 80%, or at least 90%, or substantially all of theparticles in the suspension have a diameter less than about 100 microns.

In some embodiments, the suspension formulation can comprise apharmaceutically acceptable preservative, a pharmaceutically acceptableantioxidant, or both.

In various embodiments of the suspension formulation, the structure ofthe substituted xanthine is defined. For example, in one embodiment, inreference to formula (I), R1 and R2 are the same or different and areC(1-6)alkyl or C(1-6)alkenyl; R3 is C(1-6)alkyl or hydrogen, and R4 isC(4-8) cycloalkyl. In another embodiment, R1 and R2 are the same and aremethyl or allyl, R3 is ethyl, cyclopropylmethyl or hydrogen, and R4 iscyclohexyl, provided that R1 is allyl when R3 is hydrogen, and R1 ismethyl when R3 is ethyl or cyclopropylmethyl. In another embodiment, R1and R2 are both methyl, R3 is ethyl, cyclopropylmethyl, and R4 iscyclohexyl. In another embodiment, R1 and R2 are allyl, R3 is hydrogen,and R4 is cyclohexyl, cyclohexylmethyl, or cycloheptyl. In anotherembodiment, R1 is methyl, R2 is allyl, R3 is cyclopropylmethyl or ethyl,and R4 is cyclohexyl. In yet another embodiment, R1 and R2 are the sameor different, and are methyl, propyl, allyl or hydrogen; R3 is methyl orhydrogen, and R4 is cyclohexyl or cyclopentyl. In some embodiments, thesubstituted xanthine compound is further defined, and can be1,3-dipropyl-8-cyclopentylxanthine (CPX), 1,3-diallyl-cyclohexylxanthine(DAX/DCHX), 1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX),cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In onepreferred embodiment, the substituted xanthine is1,3-dipropyl-8-cyclopentylxanthine (CPX).

The invention also provides methods for the activation of ion efflux inion efflux deficient cells. In this method, the deficient cells arecontacted, directly or indirectly, with an effective amount of a liquidsuspension suitable for oral administration, where the suspensioncomprises an effective amount of a therapeutic active ingredient,wherein said active ingredient is a substituted xanthine. Furthermore,the active ingredient is in the form of particles having a mean diameterless than about 100 microns, and the particles are in admixture with apharmaceutically acceptable oil. It is not intended that the substitutedxanthine used be particularly limited, as use of any therapeuticallyactive substituted xanthine compound, derivative of any such compound,or pharmaceutically acceptable salt of any such xanthine compound, iswithin the scope of the invention.

In a related method provided by the invention for the activation of ionefflux in ion efflux deficient cells, the substituted xanthine compoundis generally defined. In this method, the deficient cells are contacted,directly or indirectly, with an effective amount of a liquid suspensionsuitable for oral administration, where the suspension comprises aneffective amount of a therapeutic active ingredient, wherein said activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this method, the substituted xanthine is generallydefined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative. In some embodiments, the        substituted xanthine compound is further defined, and can be        1,3-dipropyl-8-cyclopentylxanthine (CPX),        1,3-diallyl-cyclohexylxanthine (DAX/DCHX),        1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX),        cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In        one preferred embodiment, the substituted xanthine is        1,3-dipropyl-cyclopentylxanthine (CPX).

In some embodiments of this methods, cells to be treated are cysticfibrosis (CF) cells, and in other embodiments, the CF cells have aCFTR-ΔF508 mutation.

In some embodiments, the pharmaceutically acceptable oil in theformulation is a vegetable oil. In some embodiments, the vegetable oilcan be corn oil, almond oil, coconut oil, cottonseed oil, mustard seedoil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybeanoil, sunflower oil, and partially or fully hydrogenated derivatives ofsaid oils.

The invention also provides methods for the activation of ion efflux inion efflux deficient cells. In this method, the deficient cells arecontacted, directly or indirectly, with an effective amount of a liquidsuspension suitable for oral administration, where the suspensioncomprises an effective amount of a therapeutic active ingredient,wherein said active ingredient is a substituted xanthine. Furthermore,the active ingredient is in the form of particles having a mean diameterless than about 100 microns, and the particles are in admixture with apharmaceutically acceptable oil. It is not intended that the substitutedxanthine used be particularly limited, as use of any therapeuticallyactive substituted xanthine compound, derivative of any such compound,or pharmaceutically acceptable salt of any such xanthine compound, iswithin the scope of the invention.

In a related method provided by the invention for the activation of ionefflux in ion efflux deficient cells, the substituted xanthine compoundis generally defined. In this method, the deficient cells are contacted,directly or indirectly, with an effective amount of a liquid suspensionsuitable for oral administration, where the suspension comprises aneffective amount of a therapeutic active ingredient, wherein said activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this method, the substituted xanthine is generallydefined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative. In some embodiments, the        substituted xanthine compound is further defined, and can be        1,3-dipropyl-8-cyclopentylxanthine (CPX),        1,3-diallyl-cyclohexylxanthine (DAX/DCHX),        1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX),        cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In        one preferred embodiment, the substituted xanthine is        1,3-dipropyl-8-cyclopentylxanthine (CPX).

In some embodiments of this methods, cells to be treated are cysticfibrosis (CF) cells, and in other embodiments, the CF cells have aCFTR-ΔF508 mutation.

In some embodiments, the pharmaceutically acceptable oil in theformulation is a vegetable oil. In some embodiments, the vegetable oilcan be corn oil, almond oil, coconut oil, cottonseed oil, mustard seedoil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybeanoil, sunflower oil, and partially or fully hydrogenated derivatives ofsaid oils.

The invention also provides methods for the activation of ion efflux inion efflux deficient cells. In this method, the deficient cells arecontacted, directly or indirectly, with an effective amount of a liquidsuspension suitable for oral administration, where the suspensioncomprises an effective amount of a therapeutic active ingredient,wherein said active ingredient is a substituted xanthine. Furthermore,the active ingredient is in the form of particles having a mean diameterless than about 100 microns, and the particles are in admixture with apharmaceutically acceptable oil. It is not intended that the substitutedxanthine used be particularly limited, as use of any therapeuticallyactive substituted xanthine compound, derivative of any such compound,or pharmaceutically acceptable salt of any such xanthine compound, iswithin the scope of the invention.

In a related method provided by the invention for the activation of ionefflux in ion efflux deficient cells, the substituted xanthine compoundis generally defined. In this method, the deficient cells are contacted,directly or indirectly, with an effective amount of a liquid suspensionsuitable for oral administration, where the suspension comprises aneffective amount of a therapeutic active ingredient, wherein said activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this method, the substituted xanthine is generallydefined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative. In some embodiments, the        substituted xanthine compound is further defined, and can be        1,3-dipropyl-8-cyclopentylxanthine (CPX),        1,3-diallyl-cyclohexylxanthine (DAX/DCHX),        1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX),        cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In        one preferred embodiment, the substituted xanthine is        1,3-dipropyl-8-cyclopentylxanthine (CPX).

In some embodiments of this methods, cells to be treated are cysticfibrosis (CF) cells, and in other embodiments, the CF cells have aCFTR-ΔF508 mutation.

In some embodiments, the pharmaceutically acceptable oil in theformulation is a vegetable oil. In some embodiments, the vegetable oilcan be corn oil, almond oil, coconut oil, cottonseed oil, mustard seedoil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybeanoil, sunflower oil, and partially or fully hydrogenated derivatives ofsaid oils.

The invention also provides methods for the activation of ion efflux inion efflux deficient cells. In this method, the deficient cells arecontacted, directly or indirectly, with an effective amount of a liquidsuspension suitable for oral administration, where the suspensioncomprises an effective amount of a therapeutic active ingredient,wherein said active ingredient is a substituted xanthine. Furthermore,the active ingredient is in the form of particles having a mean diameterless than about 100 microns, and the particles are in admixture with apharmaceutically acceptable oil. It is not intended that the substitutedxanthine used be particularly limited, as use of any therapeuticallyactive substituted xanthine compound, derivative of any such compound,or pharmaceutically acceptable salt of any such xanthine compound, iswithin the scope of the invention.

In a related method provided by the invention for the activation of ionefflux in ion efflux deficient cells, the substituted xanthine compoundis generally defined. In this method, the deficient cells are contacted,directly or indirectly, with an effective amount of a liquid suspensionsuitable for oral administration, where the suspension comprises aneffective amount of a therapeutic active ingredient, wherein said activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this method, the substituted xanthine is generallydefined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative. In some embodiments, the        substituted xanthine compound is further defined, and can be        1,3-dipropyl-8-cyclopentylxanthine (CPX),        1,3-diallyl-cyclohexylxanthine (DAX/DCHX),        1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX),        cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In        one preferred embodiment, the substituted xanthine is        1,3-dipropyl-8-cyclopentylxanthine (CPX).

In some embodiments of this methods, cells to be treated are cysticfibrosis (CF) cells, and in other embodiments, the CF cells have aCFTR-ΔF508 mutation.

In some embodiments, the pharmaceutically acceptable oil in theformulation is a vegetable oil. In some embodiments, the vegetable oilcan be corn oil, almond oil, coconut oil, cottonseed oil, mustard seedoil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybeanoil, sunflower oil, and partially or fully hydrogenated derivatives ofsaid oils.

The invention also provides methods for the treatment of a disease orcondition characterized by defective ion transport associated withreduced or abnormal CFTR activity. In this method, a subject in need isadministered a therapeutically effective amount of a liquid formulationsuitable for oral administration, where the formulation comprises aneffective amount of a therapeutic active ingredient, where the activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. It is not intended that the substituted xanthine used beparticularly limited, as use of any therapeutically active substitutedxanthine compound, derivative of any such compound, or pharmaceuticallyacceptable salt of any such xanthine compound, is within the scope ofthe invention.

In a related method provided by the invention for the treatment of adisease or condition characterized by defective ion transport associatedwith reduced or abnormal CFTR activity, the substituted xanthinecompound is generally defined. In this method, a subject in need isadministered a therapeutically effective amount of a liquid formulationsuitable for oral administration, where the formulation comprises aneffective amount of a therapeutic active ingredient, where the activeingredient is a substituted xanthine. Furthermore, the active ingredientis in the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this method, the substituted xanthine is generallydefined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative. In some embodiments, the        substituted xanthine compound is further defined, and can be        1,3-dipropyl-8-cyclopentylxanthine (CPX),        1,3-diallyl-cyclohexylxanthine (DAX/DCHX),        1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX,        cyclohexylcaffeine (CHC), or xanthine amino congener (XAC). In        one preferred embodiment, the substituted xanthine is        1,3-dipropyl-8-cyclopentylxanthine (CPX).

In some embodiments of this methods, the disease or condition to betreated is a chronic obstructive airway disorder. In another embodiment,the disease or condition to be treated is cystic fibrosis.

In some embodiments, the pharmaceutically acceptable oil in theformulation is a vegetable oil. In some embodiments, the vegetable oilcan be corn oil, almond oil, coconut oil, cottonseed oil, mustard seedoil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybeanoil, sunflower oil, and partially or fully hydrogenated derivatives ofsaid oils.

The invention also provides methods for the treatment of a disease orcondition characterized by chronic airway obstruction. In this method, asubject in need is administered a therapeutically effective amount of aliquid formulation suitable for oral administration, where theformulation comprises an effective amount of a therapeutic activeingredient, where the active ingredient is a substituted xanthine.Furthermore, the active ingredient is in the form of particles having amean diameter less than about 100 microns, and the particles are inadmixture with a pharmaceutically acceptable oil. It is not intendedthat the substituted xanthine used be particularly limited, as use ofany therapeutically active substituted xanthine compound, derivative ofany such compound, or pharmaceutically acceptable salt of any suchxanthine compound, is within the scope of the invention.

In a related method provided by the invention for the treatment of adisease or condition characterized by chronic airway obstruction, thesubstituted xanthine compound is generally defined. In this method, asubject in need is administered a therapeutically effective amount of aliquid formulation suitable for oral administration, where theformulation comprises an effective amount of a therapeutic activeingredient, where the active ingredient is a substituted xanthine.Furthermore, the active ingredient is in the form of particles having amean diameter less than about 100 microns, and the particles are inadmixture with a pharmaceutically acceptable oil. In this method, thesubstituted xanthine is generally defined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen. Also encompassed by        this method is use of therapeutically active derivatives of the        substituted xanthine, pharmaceutically acceptable salt of the        substituted xanthine, and pharmaceutically acceptable salt of        the therapeutically active derivative.

The present invention also provides articles of manufacture comprisingthe formulations of the invention. In one embodiment, the article ofmanufacture provides a container, a liquid pharmaceutical formulationsuitable for oral administration, comprising a therapeutic activeingredient, wherein said active ingredient is a substituted xanthine,and directions for the administration of the formulation for thetreatment of a disease or condition characterized by defective iontransport associated with reduced or abnormal CTFR activity.Furthermore, the active ingredient is in the form of particles having amean diameter less than about 100 microns, and the particles are inadmixture with a pharmaceutically acceptable oil. It is not intendedthat the substituted xanthine used in the article of manufacture beparticularly limited, as use of any therapeutically active substitutedxanthine compound, derivative of any such compound, or pharmaceuticallyacceptable salt of any such xanthine compound, is within the scope ofthe invention.

In a related composition, the present invention also provides articlesof manufacture comprising the formulations of the invention as describedabove, and the substituted xanthine compound is generally defined. Inone embodiment, the article of manufacture provides a container, aliquid pharmaceutical formulation suitable for oral administration,comprising a therapeutic active ingredient, wherein said activeingredient is a substituted xanthine, and directions for theadministration of the formulation for the treatment of a disease orcondition characterized by defective ion transport associated withreduced or abnormal CTFR activity. Furthermore, the active ingredient isin the form of particles having a mean diameter less than about 100microns, and the particles are in admixture with a pharmaceuticallyacceptable oil. In this embodiment, the substituted xanthine isgenerally defined by the formula:

-   -   (I), wherein    -   R1 and R2 are the same or different and are C(1-6)alkyl or        C(1-6)alkenyl, or hydrogen; R3 is C(1-6)alkyl or hydrogen, and        R4 is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one        of R1, R2 and R3 is other than hydrogen, or a therapeutically        active derivative thereof, or a pharmaceutically acceptable salt        of said substituted xanthine, or a pharmaceutically acceptable        salt of said therapeutically active derivative.

In other embodiments of the article of manufacture, the instructions arein the form of a package insert. In other embodiments, the disease orcondition to be treated is cystic fibrosis.

In another embodiment of the article of manufacture, the container is abottle. In another embodiment, the bottle is a glass bottle. In stillanother embodiment, the glass bottle is secured by a cap.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of rat plasma CPX concentration (ng/ml) as afunction of time (in hours) using two different drug formulations. TheCPX concentration was measured at regular time intervals following oraladministration of an approximately 100 mg dose, where the dose wasdelivered in either a corn oil formulation (dark line) or a waterformulation (light line).

FIG. 2 shows a table of CPX concentrations in the blood plasma of fourmale Beagle dog at regular time intervals (shown in hours) following theadministration single oral doses 30 mg/kg of various CPX formulations.These formulations were xanthan gum (homogenized), sodiumcarboxymethylcellulose [NaCMC] (homogenized), sodiumcarboxymethylcellulose [NaCMC] (non-homogenized), and corn oil(homogenized).

FIG. 3 shows a graphical representation of the data provided in FIG. 3,where the mean CPX plasma concentration (ng/ml) of each dog treatmentgroup is plotted against time (in hours), for each drug formulation.This representation plots the mean plasma CPX concentrations on a linearaxis.

FIG. 4 shows a graphical representation of the data provided in FIG. 3,where the mean CPX plasma concentration (ng/ml) of each dog treatmentgroup is plotted against time (in hours), for each drug formulation.This representation plots the mean plasma CPX concentrations on asemilogarithmic axis.

FIG. 5 shows human blood plasma CPX concentrations at regular timeintervals (in hours) following administration of a single 300 mg oraldose of CPX. Data for two human subject groups is shown, one group (n=3)receiving the CPX dose in a corn oil formulation (diamonds), and theother group (n=4) receiving the CPX dose in a hard gelatin capsule(triangles). Standard error of the mean for each time point is shown asa vertical line.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For further information see,for example, Comprehensive Organic Chemistry, I. O. Sutherland editor,Pergamon Press, 1979; Vogel's Textbook of Practical Organic Chemistry,5th Ed., 1989; Van Nostrand Reinhold, Encyclopedia of Chemistry, 4thEd., 1984; John McMurry, Organic Chemistry, 5th Ed., 2000; Vollhardt andSchore, Organic Chemistry, W.H. Freeman and Co., New York, 1995. Oneskilled in the art will recognize many methods and materials similar orequivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

The term “suspension” is used for its ordinary meaning to describe adispersion of solid particles in a liquid. Thus, an “oil-basedsuspension” means the suspension of solid particles in an oil.

The term “homogenized” or “homogenous” is used to refer to asubstantially uniform distribution of solid particles (e.g., drugparticles) in a suspension, such as an oil-based suspension.

The term “liquid formulation” is used to describe any mixture of two ormore substances which is substantially liquid in character. Liquidformulations include, without limitation, solutions, suspensions anddispersions of an active ingredient, and optionally further components,in a liquid excipient, preferably an oil, such as a vegetable oil.Liquid formulations, as defined herein, can comprise both particulateand dissolved components. Furthermore, the liquid formulations hereincan comprise the same component in both particulate and dissolved form.

“Particle size distribution” means the number of particles in individualsize classes divided by the total number of particles in a sample,expressed as percentages. Particle size distribution can be determinedby a variety of techniques known in the art, such as quantitativemicroscopic examination, or laser diffraction methodology. A preferredmethod is laser diffraction analysis (also called low angle lightscattering), by which dry powders can be measured directly and liquidsuspensions and emulsions can be measured in a re-circulating cell. Thisgives high reproducibility and enables the use of dispersing agents andsurfactants for the determination of primary particle size. Particlesize analyzers are commercially available, for example from BeckmanCoulter, U.S., Laval Lab Inc., Canada, and Malvern Instruments Ltd.,USA, the manufacturer of a variety of Mastersizer® particle analyzers.

A suspension in which “substantially all” particles has a diameter lessthan 100 microns contains at least about 95%, more preferably at leastabout 98%, even more preferably at least about 99%, most preferably atleast about 99.5% particles with a diameter less than about 100 microns.

The “pharmaceutically acceptable oil” can be any natural or syntheticvegetable or animal oil suitable for pharmaceutical use, comprisingmono-, di-, or triglyceryl esters of saturated and/or unsaturated fattyacids, alone or in combination.

The term “mammal” or “mammalian species” refers to any animal classifiedas a mammal, including humans, domestic and farm animals, and zoo,sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs,goats, rabbits, as well as rodents such as mice and rats, etc.Preferably, the mammal is human.

The terms “subject” or “patient,” as used herein, are usedinterchangeably, and can refer to any to animal, and preferably amammal, that is the subject of an examination, treatment, analysis, testor diagnosis. In one embodiment, humans are a preferred subject. Asubject or patient may or may not have a disease or other pathologicalcondition.

The terms “disease,” “disorder” and “condition” are used interchangeablyherein, and refer to any disruption of normal body function, or theappearance of any type of pathology. The etiological agent causing thedisruption of normal physiology may or may not be known. Furthermore,although two patents may be diagnosed with the same disorder, theparticular symptoms displayed by those individuals may or may not beidentical.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the objective is toprevent or slow down (lessen) an undesired physiological change ordisorder. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. Those in need of treatmentinclude those already with the condition or disorder as well as thoseprone to have the condition or disorder or those in which the conditionor disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain a desiredeffect or level of agent(s) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is periodic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic (including preventative) results. An effectiveamount can be administered in one or more administrations.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “alkyl” refers to a monovalent alkane (hydrocarbon) derivedradical containing from 1 to 10 carbon atoms unless otherwise defined.It may be straight- or branched-chained, or cyclic. Preferred straight-or branched-chained alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, and t-butyl. Preferred cycloalkyl groups includecyclopropyl, cyclobutyl, cycloheptyl, cyclopentyl, and cyclohexyl. Theterm “lower alkyl” refers to alkyl groups as hereinabove defined, having1 to 6 carbon atoms. The term “alkyl” as used herein includessubstituted alkyls.

The term “substituted alkyl” refers to alkyl as defined above, includingone or more functional groups such as lower alkyl, aryl, acyl, halogen,hydroxy, amino, alkoxy, alkylamine, acylamino, acyloxy, aryloxy,aryloxyalkyl, mercapto, both saturated and unsaturated cyclichydrocarbons, heterocycles, and the like. These groups may be attachedto any carbon of the alkyl moiety.

The term “aryl” is used herein to refer to an aromatic substituent whichmay be a single aromatic ring or multiple aromatic rings which are fusedtogether, linked covalently, or linked to a common group such as amethylene or ethylene moiety. The common linking group may also be acarbonyl as in benzophenone. The aromatic ring(s) may include phenyl,naphthyl, biphenyl, diphenylmethyl and benzophenone among others. Theterm “aryl” encompasses “arylalkyl,” “arylalkenyl,” and “arylalkinly.”The term “aryl” as used herein also includes substituted aryl.

“Substituted aryl” refers to aryl, as defined above, including one ormore functional groups such as lower alkyl, acyl, halogen, alkylhalo,hydroxy, amino, alkoxy, alkylamine, acylamino, acyloxy, mercapto andboth saturated and unsaturated cyclic hydrocarbons which are fused tothe aromatic ring(s), linked covalently or linked to a common group suchas a methylene or ethylene moiety. The linking group may also be acarbonyl such as in cyclohexyl phenyl ketone. The term “substitutedaryl” encompasses “substituted arylalkyl.”

The term “aralkyl” or “arylalkyl” is used to refer to an aryl orheteroaryl moiety, as defined herein, attached through a C1-6 alkyllinker, where alkyl is as defined above.

The term “alkoxy” refers to a substituent with a straight- orbranched-chain alkyl, alkenyl, or alkinyl group of the designatedlength, which is attached via an oxygen molecule. Representative alkoxygroups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,t-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy, allyloxy,propargyloxy, vinyloxy, etc.

The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

The term “amino” is used to refer to the group —NRR′, where R and R′ mayindependently be hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl or acyl.

The term “alkoxy” is used herein to refer to an —OR group, where R is alower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkylor substituted arylalkyl wherein the alkyl, aryl, substituted aryl,arylalkyl and substituted arylalkyl groups are as described herein.Suitable alkoxy radicals include, for example, methoxy, ethoxy, phenoxy,substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.

The term “alkenyl” is used herein to refer to an unsaturated straight-or branched-chained, or cyclic monovalent hydrocarbon radical having atleast one carbon-carbon double bond. The radical can be in either thecis or trans conformation about the double bond(s). Suitable alkenylradicals include, for example, ethenyl, propenyl, isopropenyl,cyclopropenyl, butenyl, isobutenyl, cyclobutenyl, tert-butenyl,pentenyl, hexenyl, etc.

The term “pharmaceutically acceptable salt” refers to those salts ofcompounds which retain the biological effectiveness and properties ofthe free bases and which are obtained by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. Pharmaceuticallyacceptable salts include, for example, alkali metal salts, such assodium and potassium, alkaline earth metal salts and ammonium salts.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides novel, improved drug formulationscomprising xanthine derivatives, where the novel formulations haveimproved characteristics such as drug uptake and bioavailability. Thesenovel formulations find use in the treatment of various diseases,including but not limited to diseases resulting from defective iontransport due to reduced or abnormal CFTR activity, such as in cysticfibrosis, and also more broadly in the treatment of COPD. It is alsocontemplated that these formulations find use in the treatment of otherdiseases resulting from or exacerbated by improper-ion balances.

Xanthine Compounds Finding Use in the Treatment of Cystic Fibrosis orOther Diseases

Numerous xanthine derivatives are known to have properties consistentwith therapeutic value in the treatment of cystic fibrosis and otherdiseases. Such xanthine derivatives include those characterized by thefollowing general formula (I)

-   -   wherein R1 and R2 are the same or different and are C(1-6)alkyl,        C(1-6)alkenyl or hydrogen; R3 is C(1-6)alkyl or hydrogen, and R4        is C(4-8)cycloalkyl, aryl or hydrogen, wherein at least one of        R1, R2 and R3 is other than hydrogen, and therapeutically active        derivatives thereof, or pharmaceutically acceptable salts of        such compounds or their derivatives.

In a preferred embodiment, R1 and R2 are the same or different and areC(1-6)alkyl or C(1-6)alkenyl; R3 is C(1-6)alkyl or hydrogen, and R4 isC(4-8) cycloalkyl.

In another preferred embodiment, R1 and R2 are the same and are methylor allyl, R3 is ethyl, cyclopropylmethyl or hydrogen, and R4 iscyclohexyl, provided that R1 is allyl when R3 is hydrogen, and R1 ismethyl when R3 is ethyl or cyclopropylmethyl.

In yet another preferred embodiment, the formulation comprises acompound of formula (I) in which R1 and R2 are both methyl, R3 is ethylor cyclopropylmethyl, and R4 is cyclohexyl.

In other preferred compounds, R1 and R2 are allyl, R3 is hydrogen; andR4 is cyclohexyl, cyclohexylmethyl, or cycloheptyl; or R1 is methyl, R2is allyl, R3 is cyclopropylmethyl or ethyl, and R4 is cyclohexyl.

In still other preferred embodiments, the formulation comprises acompound of formula (I), wherein R1 and R2 are the same or different andare methyl, propyl, allyl or hydrogen; R3 is methyl or hydrogen, and R4is cyclohexyl or cyclopentyl, and therapeutically active derivativesthereof, or pharmaceutically acceptable salts of such compounds or theirderivatives.

The xanthine derivatives used in the formulations of the presentinvention can be synthesized by standard methods of organic chemistry,such as those described in the textbooks referenced above, and alsoe.g., in Jacobson et al., Biochemistry 34:9088-94 (1995); and U.S. Pat.Nos. 6,248,746; 6,180,791; 5,981,535; 5,366,977, 5,877,179 and6,083,954. Alternatively, the compounds are commercially available(e.g., from Research Biochemicals International [RBI/Sigma], Natick,Me., and Sigma-Aldrich, St. Louis, Mo.).

Assays to identify xanthine derivatives, others than specificallydisclosed herein, potentially useful for the treatment of cysticfibrosis and other diseases associated with reduced apicalCl—conductance in cells, are known in the art. For example, known drugscreening assays for the identification of further useful xanthinederivatives include:

(A) Chloride Efflux Assay using Recombinant CFTR—Normal culturedmammalian cells, and most preferably human cells, are transfected withan expression vector encoding the wild-type or ΔF508 CFTR gene product.While in culture, the cells are treated with drug candidate compounds,and the chloride efflux across the cell membranes is measured, forexample, by radiolabelled chloride isotopic equilibrium. Alternatively,changes in the osmolarity of the cell external medium can also bemeasured using an osmometer. This technique (or variations thereof) aredescribed in various sources, such as but not limited to, U.S. Pat. No.6,083,954; and Eidelman et al., Proc. Nail. Aced Sc. USA 89:5562-5566[1992]. Compounds that stimulate chloride efflux in vitro are candidatedrugs for further development and testing.

(B) Chloride Efflux Assay using Native Mutant CFTR—Similar to thetechnique described above, cultured mammalian cells, and mostpreferably, human cells derived from a cystic fibrosis patient (i.e.,primary explant cultures), and most preferably where the cells arehomozygous for the CFTR-ΔF508 mutation, are treated with drug candidatecompounds, and the chloride efflux across the cell membrane is measured.For example, this technique (or variations thereof are described inEidelman et al., supra.

(C) CFTR-Protein Binding Assay—Purified wild-type CFTR or mutant CFTR(e.g., CFTR-ΔF508) protein, or suitable portions thereof, can beutilized in vitro to identify compounds (i.e., drug candidates) thathave the ability to bind CFTR in a protein-specific manner and with highaffinity. Methods for the determination and quantitation of proteinbinding specificity and binding affinity are known in the art. Thebinding can be by any particular manner, but is most typically bynon-covalent forces, such as hydrogen bonding, adsorption, absorption,metallic bonding, van der Waals forces, ionic bonding, or anycombination thereof. In this case, portions of CFTR comprising the firstnucleotide binding fold (NBF-1) are the preferred portions of CFTR touse in this type of assay. Compounds that bind with high affinity toCFTR, or a suitable portion of CFTR, are candidates for furtherdevelopment and testing.

Alternatively, the ability of a compound to bind to the wild-type andmutant CFTR proteins can be compared to identify candidate drugs, wherecompounds that bind preferentially to CFTR-ΔF508 compared to wild-typeCFTR are also candidates for further development and testing.

The identification of compounds with binding specificity for CFTRprotein, where the compound does not bind or binds with less affinity toother proteins, is a valuable indicator for drug screening. This isespecially significant with regard to adenosine receptor proteins. Somecompounds have been shown to bind the A1, A2 or A3-adenosine receptors,and/or antagonize the activity of those receptors. Compounds thatantagonize adenosine receptors may not be ideal candidates for drugdevelopment, as those compounds may have toxic side effects whenadministered to a subject. However, it is not intended that the xanthinecompounds finding use with the invention are limited to those compoundsthat do not bind or otherwise do not antagonize an adenosine receptor.

(D) Biochemical Activity Assays—Purified CFTR protein, or suitableportions of the protein, can be assayed in vitro for various biochemicalactivities in the absence and presence of drug candidate compounds. Theinduction or suppression of these activities in response to exposure toa test compound may be indicative that the compound has advantageoususes in the treatment of cystic fibrosis. For example, the various invitro CFTR activities that can be measured include commencing or causingthe aggregation of bovine chromaffin granules in the presence of CaCl₂,and commencing or causing the aggregation of liposomes. Such assays aredescribed, for example, in U.S. Pat. No. 6,083,954.

It is not intended, however, that the present invention be limited toformulations comprising substituted xanthine compounds that strictlyadhere to the above screening criteria. Furthermore, it is not intendedthat the invention be limited to any particular biochemical mechanism,as an understanding of the biochemical mechanisms underlying theproperties of the invention is not necessary to make or use theinvention. Thus, it is not necessary to have any understanding of themechanism of the invention to make or use the invention.

It is intended, without limitation, that the substituted-xanthinecompounds taught in U.S. Pat. Nos. 5,366,977, 5,877,179 and 6,083,954,the disclosures of which are hereby incorporated by reference in theirentirety, find use in the novel drug formulations of the presentinvention.

Specific xanthine derivatives which find use in formulations of thepresent invention are listed below. However, it is not intended that thepresent invention be limited to those compounds listed below, as one ofskill in the art immediately recognizes that variant molecules withstructures related to the structures of the molecules listed below alsofind use with the invention.

-   1,3-dipropyl-7-methyl-8-cyclohexyl-xanthine-   1,3-dipropyl-7-methyl-8-cyclopentyl-xanthine (DP-CPX)-   1,3-diallyl-8-cyclohexyl-xanthine (DCHX)-   1,3-dipropyl-8-cyclopentyl-xanthine (CPX)-   1-propyl-8-cyclopentyl-xanthine-   N,N-diallyl-8-cyclohexyl-xanthine (DAX)-   1,3-diallyl-8-cyclohexyl-xanthine DCHX-   1,3-dipropyl-7-methyl-8-cyclohexyl-xanthine-   8-cyclohexyl caffeine (1,3,7-trimethyl-8-cyclohexyl-xanthine; CHC)-   1,3-dimethyl-8-cyclohexyl-xanthine-   1,3,7-trimethyl-8-(3-chlorostyrl)-xanthine (aka CSC)-   theophylline-   IBMX-   xanthine amino congener (XAC)-   KFM19-   2-thio-CPX-   KW-3902-   CPT-   caffeine

These compounds are described in various sources, including but notlimited to, U.S. Pat. Nos. 4,548,818, 4,866,072, 5,032,593, 5,096,916,5,366,977, 5,877,179 and 6,083,954; and references such as von der Leyenet al., Naunyn Schmiedebergs Arch. Pharmacol., 340(2):204-209 [1989];Guay-Broder et al., Biochemistry 34(28):9079-9087 (1995); Müller et al.,J. Med. Chem., 36:3341-3349 [1992]; Jacobson et al., Biochemistry34:9088-9094 [1995]; Roomans, Exp. Opin. Invest Drugs 10(1):1-19 [2001];Arispe et al., Jour. Biol. Chem., 273(10):5727-5734 [1998]; Rogers andKnox, Eur. Respir. Jour., 17:1314-1321 [2001]; Eidelman et al., Proc.Natl. Aced. Sc. USA 89:5562-5566 [1992]; and Ji et al., Jour. ReceptorResearch 12(2):149-169 [1992]. It is contemplated that substitutedxanthine compounds described in the above U.S. patents and referencescan find use in the formulations of the invention.

Particularly preferred xanthine derivatives for use in the formulationsof the present invention are 1,3-dipropyl-8-cyclopentylxanthine (CPX),N,N-diallylcyclohexylxanthine (DAX; synonymously,1,3-diallyl-8-cyclohexylxanthine, DCHX),1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX), cyclohexylcaffeine(CHC), and xanthine amino congener.

Alternatively, or additionally, a pharmaceutically acceptable derivativeof any of the compounds of the invention, or combinations of compounds,may be used in the present invention and inventive method, which provideyet another embodiment of the present invention. It is desirable thatsuch a pharmaceutical derivative have equivalent therapeuticeffectiveness in the context of the present inventive method oftreatment.

In a most preferred embodiment, the compound1,3-dipropyl-8-cyclopentyl-xanthine (CPX) is used in the formulations ofthe invention, which is in clinical development for the treatment ofcystic fibrosis. CPX has numerous advantageous properties, including butnot limited to, (a) activates chloride efflux from cell derived from acystic fibrosis patient, (b) activates isolated CFTR channels in invitro lipid bilayers, (c) binds to the NBF-1 region of CFTR, (d) itsaffinity for CFTR-ΔF508 NBF-1 is greater than the affinity of CPX forwild-type CFTR NBF-1, (e) enhances intracellular trafficking andmaturation of CFTR-ΔF508, and (f) does not appear to display anymutagenicity or grossly apparent adverse side effects when oral dosesare delivered to rat, guinea pig, mouse or dog model systems.Furthermore, CPX shows no apparent adverse side effects when oral dosesare delivered to human subjects.

The present invention also encompasses all pharmaceutically acceptablesalts of the foregoing compounds. One skilled in the art will recognizethat acid addition salts of the presently claimed compounds may beprepared by reaction of the compounds with the appropriate acid via avariety of known methods. Alternatively, alkali and alkaline earth metalsalts are prepared by reaction of the compounds of the invention withthe appropriate base via a variety of known methods. For example, thesodium salt of the compounds of the invention can be prepared byreacting the compound with sodium hydride.

In the formulations of the present invention, the compounds of formula(I), including their derivatives and salts, are in pharmaceuticallyacceptable form. By pharmaceutically acceptable form is meant, interalia, a pharmaceutically acceptable level of purity excluding normalpharmaceutical additives such as diluents and carriers, and including nomaterial considered toxic at normal dosage levels. A pharmaceuticallyacceptable level of purity will generally be at least about 50%excluding normal pharmaceutical additives, preferably at least about75%, more preferably at least about 90% still more preferably at leastabout 95%, and most preferably at least about 98%.

Preferably, the active compounds of formula (I) are sterilized beforeincorporation into the suspension formulations of the present invention.Sterilization may be performed, for example, by exposure to ethyleneoxide before incorporation into the sterile vehicle (e.g., an oil).

Oil-based Suspensions of Substituted Xanthine Compounds for OralAdministration

Known formulations for the therapeutic delivery of substituted xanthinecompounds utilize an aqueous delivery vehicle. In an effort to identifyimproved drug formulations displaying more advantageous pharmacokineticproperties, such as bioavailability and plasma half-life, the presentinventors developed novel, oil-based formulations of substitutedxanthine compounds suitable for oral delivery. More specifically,suspensions of xanthine derivatives in pharmaceutically acceptable oilswith improved bioavailability and pharmacokinetic properties have beendeveloped.

In tests that have led to the present invention, suspensions of CPX incorn oil were tested. In addition to CPX, this suspension formulationcontained methylparaben and propylparaben as preservatives, andbutylated hydroxytoluene (BHT) as an antioxidant. Details describing thepreparation of the formulation are provided in Experimental EXAMPLE 1.

This corn oil-based formulation was used in side-by-side analyses withother CPX formulations, such a formulations comprising water, sodiumcarboxymethylcellulose (NaCMC; in homogenized or non-homogenizedformulations), xanthan gum, and/or gelatin capsules, to testpharmacokinetic properties and bioavailability in rat and dog modelsystems. The corn oil-based formulations consistently showedstatistically significant improved properties compared to otherformulations.

For example, as described in Experimental EXAMPLE 2, the blood plasmadrug concentration of CPX was determined in rats following oraladministration using either a water (i.e., aqueous) or a corn oil CPXformulation. As can be seen in FIG. 1, the differences in systemic CPXconcentrations between the water and corn oil formulations is striking.The corn oil group displayed significant and sustained plasma CPX aslong as 8 hours following drug delivery, while no individuals in thewater vehicle group displayed any detectable plasma CPX.

Experimental EXAMPLE 3 describes additional advantageous properties ofcorn oil formulations using a dog model system. Using this model system,the pharmacokinetics of CPX absorption were measured using various oralCPX formulations, including xanthan gum (homogenized), sodiumcarboxymethylcellulose [NaCMC] (homogenized), sodiumcarboxymethylcellulose [NaCMC] (non-homogenized), and corn oil(homogenized) formulations. In this experiment, the pharmacokineticparameters C_(max) (maximum analyte concentration in the plasma, ng/ml),T_(max) (time of maximum analyte concentration in the plasma), and AUC(area under the curve for a defined period of time, where AUC is ameasure of total systemic exposure expressed as ng-h/ml). The results ofthis experiment are summarized in FIGS. 2 and 3, and in TABLE 4. As canbe seen in these FIGS. and TABLE, oral administration of the corn oilsuspension formulation resulted in systemic CPX exposure which was atleast two-fold greater than any other formulation tested. Based onplasma AUC(O-8) and C_(max) comparisons of the formulations tested, theoral bioavailability was highest with the corn oil formulation. Thus,the use of a corn oil CPX delivery formulation results in greatermaximal drug concentration and greater overall systemic drug exposurecompared to any other formulation tested.

Human clinical studies were also undertaken to test the pharmacokineticproperties of an orally administered standard gelatin capsule CPXformulation or a novel corn oil CPX formulation. These pharamcokineticproperties were determined by monitoring the blood plasma CPXconcentrations following oral administration of the formulations. Thesestudies are described in Experimental EXAMPLE 4, and results are shownin FIG. 5 and TABLE 5. As can be seen in this data, the CPXconcentrations in the subjects receiving the corn oil formulation reacha statistically significant higher level, and reach a C_(max) value muchquicker compared to the concentration values in the subjects receivingthe gelatin capsule CPX formulation. The corn oil formulation of CPXprovided at least a two-fold greater maximal plasma CPX concentration,at least double total systemic CPX exposure (as measured by AUC_(inf)),and a longer half-life of the drug in the blood plasma (as measured byT_(1/2))

Although corn oil is used as an exemplary oral drug delivery vehicle, itis not intended that the present invention be limited exclusively to theuse of corn oil as the drug delivery vehicle for substituted xanthinecompounds. It is contemplated that numerous pharmaceutically acceptablenatural or synthetic vegetable or animal oils find use as the oral drugdelivery vehicle. Thus, oils suitable for use in the formulations of thepresent invention include vegetable oils, fish oils, animals fats andtheir partially or fully hydrogenated derivatives.

In a preferred embodiment, the delivery vehicle is a natural orsynthetic pharmaceutically acceptable vegetable oil, comprising mono-,di, and/or trilgyceryl esters of saturated and/or unsaturated fattyacids. It is preferred that the oil be a glyceryl ester of a C₁₄-C₂₂saturated and/or unsaturated fatty acids, triglycerides beingparticularly preferred.

Exemplary vegetable oils suitable for use as delivery vehicles in theformulations of the present invention include aceituno oil, almond oil,araehis oil, babassu oil, blackcurrant seed oil, borage oil, buffaloground oil, candlenut oil, canola oil, caster oil, Chinese vegetabletallow oil, cocoa butter, coconut oil, coffee seed oil, corn oil,cottonseed oil, crambe oil, Cuphea species oil, evening primrose oil,grapeseed oil, groundnut oil, hemp seed oil, illipe butter, kapok seedoil, linseed oil, menhaden oil, mowrah butter, mustard seed oil,oiticica oil, olive oil, palm oil, palm kernel oil, peanut oil, poppyseed oil, rapeseed oil, rice bran oil, safflower oil, sal fat, sesameoil, shark liver oil, shea nut oil, soybean oil, stillingia oil,sunflower oil, tall oil, tea seed oil, tobacco seed oil, tung oil (Chinawood oil), ucuhuba, vernonia oil, wheat germ oil, hydrogenated casteroil, hydrogenated coconut oil, hydrogenated cottonseed oil, hydrogenatedpalm oil, hydrogenated soybean oil, hydrogenated vegetable oil,hydrogenated cottonseed and caster oil, partially hydrogenated soybeanoil, partially hydrogenated soy and cottonseed oil, glyceryltributyrate, glyceryl tricaproate, glyceryl tricaprylate, glyceryltricaprate, glyceryl trundecanoate, glyceryl trilaurate, glyceryltrimyristate, glyceryl tripalmitate, glyceryl tristearate, glyceryltriarcidate, glyceryl trimyristoleate, glyceryl tripalmitoleate,glyceryl trioleate, glyceryl trilinoleate, glyceryl trilinolenate,glyceryl tricaprylate/caprate, glyceryl tricaprylate/caprate/laurate,glyceryl tricaprylate/caprate/linoleate, glyceryltricaprylate/caprate/stearate, glyceryl tricaprylate/laurate/stearate,glyceryl 1,2-caprylate-3-linoleate, glyceryl 1,2-caprate-3-stearate,glyceryl 1,2-laurate-3-myristate, glyceryl 1,2-myristate-3-laurate,glyceryl 1,3-palmitate-2-butyrate, glyceryl 1,3-stearate-2-caprate;glyceryl 1,2-linoleate-3-caprylate.

Vegetable and non-vegetable oils, e.g., oils of animal origin, suitablefor use in pharmaceutical formulations, as listed above, are readilyavailable from commercial sources, including for example, Croda, Inc.and Croda International Plc. (East Yorkshire, UK), Abitec Corporation(London, UK and Columbus, Ohio), Research Plus, Inc. (South Plainfield,N.J.), Sigma (St. Louis, Mo.) and Larodan Fine Chemicals (Malmö,Sweden).

In some embodiments, a particularly noteworthy advantage of theinvention is realized when the vegetable oil drug delivery formulationis used to deliver a substituted xanthine therapeutic compound that iswater-sensitive and/or unstable in aqueous formulations, therebyprotecting the drug from degradation.

In some embodiments, the vegetable oil formulations of the inventioncontain only vegetable oil and the xanthine drug. In other embodiments,the vegetable oil formulation comprises additional components such aspreservatives (e.g., methylparaben and/or propylparaben), antioxidants(e.g., butylated hydroxytoluene; BHT), thickening agents, sweeteners(e.g. sucrose; lactose, fructose, glucose, mannitol, sorbitol,saccharin, cyclamate, acesulfam potassium, or taumatin), bufferingagents, surfactants, solubilizers, flavorings (e.g., raspberry,strawberry and honey), odorants and/or colorants.

The pharmaceutical formulations of the present invention are provided inthe form of oil-based suspensions, and intended for oral administration,optionally followed by the consumption of water. Accordingly, theconcentration of the xanthine derivative in the formulation may varywithin a wide range, and can preferably be up to the maximum amount thatcan be suspended, and further, the xanthine derivative has a homogenousuniform and optimal particle size that is instrumental in increasingbioavailability In general, the concentration of the xanthine derivativewill be between about 0.1% and about 50% by weight, more preferablybetween about 1% and about 20% by weight, more preferably between about1% and about 10% by weight.

A preferred pharmaceutical formulation herein has the followingcomposition: Concentration Range Component (% by weight) vegetable oil85-95 preservative 0.0-0.5 antioxidant 0.0-0.5 xanthine derivative  1-10

In a particularly preferred embodiment, the formulations contain about90-95% by weight corn oil, 4.0-8.0% by weight xanthine derivative, e.g.,CPX, 0.05 to 0.15% by weight methylparaben and/or propylparaben, andoptionally 0.05 to 0.15% butylated hydroxytoluene.

For optimal bioavailability, the mean particle size of the xanthine drugparticle dispersed in the formulations should be less than about 100microns. In one embodiment of the invention, the drug particles arepreferably less than about 80 microns. In another embodiment, the drugparticles are less than about 70 microns. In another embodiment, thedrug particles are less than about 65 microns. However, it is notintended that the invention be limited to any particular drug particlesize less than about 100 microns. It is contemplated that a range ofparticle sizes are equally suitable for use in the drug formulations.Furthermore, it is contemplated that different methods for drugcrystallization will result in drug particles having differing and/ormore advantageous properties. Different methods for drug crystallizationcan result in different optimal drug particle sizes to be used in theformulations. Thus, it is not intended that the invention be limited toany particular method for drug synthesis or crystallization, or drugparticle size or size range.

If one round of homogenization does not provide the desired particlesize and distribution, the homogenization process is repeated to ensurethat the drug-particles are of the desired diameter. The small particlesize of the active substance in the dispersions of the present inventionas described also has the advantage of a slow rate of sedimentation ofthe suspended particles, which favorably affects the homogeneity of theliquid oral formulation of the active substance described andcorrespondingly ensures a high degree of accuracy in measuring the dose.

Preferably the formulations of the present invention are suitable forlong term storage, and remain stable at room temperature for at least 6months.

The formulations can be packaged into conventional containers, such asplastic or glass bottles conventionally used in the drug industry. Thebottles are typically secured by a plastic screw cup, which ispreferably child-resistant, have a label affixed to them, and might beaccompanied by written directions for administration. Such articles ofmanufacture are within the scope of the invention.

The compound should be administered such that a therapeuticallyeffective concentration of the compound is in contact with the affectedcells of the body. The dose administered to a subject, particularly ahuman, in the context of the present invention should be sufficient toeffect a therapeutic response in the animal over a reasonable period oftime. The dose will be determined by the strength of the particularcompound employed and the condition of the subject, as well as the bodyweight of the animal to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particularcompound.

The following examples serve to further illustrate the present inventionand are not intended to limit the scope of the invention.

1. EXAMPLE 1

(a) CPX Formulations

This EXAMPLE describes the CPX formulations used in the presentinvention. Adequate solubility of CPX was unattainable in any of thesolvents tested, even despite the use of co-solvents. This insolubilitynecessitated the use of the suspension and capsule formulations, asdescribed below. One formulation was left non-homogenized to study theeffect of homogenization on bioavailability.

Homogenization of CPX liquid formulations by mechanical means was usedto attain uniform small particle size and a homogenous suspension. Thedrug/liquid vehicle mixtures were homogenized using a Brinkmann PolytronPT 6000 Homogenizer with the PT-DA 6045/6T generator at a homogenizationspeed setting of 10,800 rpm. Homogenization resulted in a mean particlediameter size of approximately 65 μm.

The formulations were prepared with various concentrations of CPXdepending on the intended experiment, the intended model organism to bestudied, or whether is would be used for human trials. The concentrationof CPX in these formulations was confirmed using high performance liquidchromatography (HPLC) with mass spectrometric determination. Theformulations were produced using Good Manufacturing Procedures (cGMPs).Quality assurance testing showed the formulations to be within theintended specification and sufficiently sterile. All formulations werestored at room temperature, and were demonstrated to be stable for atleast 3 months. The formulations used were:

A) Sodium Carboxymethylcellulose [NaCMC] (homogenized), 2.175%. Themixture was homogenized to form a suspension, as described above.

B) Sodium Carboxymethylcellulose [NaCMC] (not homogenized), 2.175%

C) Xanthan Gum (homogenized), 0.4%.

The xanthan gum formulation used herein was a polysaccharide mixturecontaining glucose, mannose, potassium glucuronate, acetate andpyruvate. A drug delivery vehicle was formed by producing an aqueous0.4% xanthan gum suspension, then adding CPX drug to a suitableconcentration. The mixture was homogenized to form a suspension, asdescribed above.

D) Corn Oil (homogenized)

This formulation used corn oil as the delivery vehicle for CPX drug. Inaddition to the CPX drug, this suspension formulation containedmethylparaben and propylparaben as preservatives, and butylatedhydroxytoluene (BHT) as an antioxidant.

Specifically, 9.853 kg of corn oil precombined with BHT was placed in a20 liter mixing vessel and mixed at a speeds ranging from 444 to 750 RPMduring the mixing process. To the stirring corn oil, 10.50 grams (0.1%by weight) of methylparaben was added, and stirred for 47 minutes untildissolved. Once dissolved, 6.3 grams (0.06% by weight) propylparabenwere added to the corn oil mixture, and stirred for 73 minutes untildissolved. To this was then added 630 grams (6.0% by weight) CPX, andstirred for 40 minutes until the CPX was uniformly dispersed. Thismixture was homogenized to form a suspension, as described above.

Placebo is supplied as corn oil solution containing methylparaben,propylparaben and butylated hydroxytoluene (BHT). Each dose of placebomatches the dose weight and volume utilized in the active portion of thecorresponding non-placebo administration.

In one case, the CPX-corn oil formulation was packaged in a vesselconvenient for dispensing the formulation to a subject. The vessel was a2 ounce (capacity approximately 60 ml) amber glass bottle secured with achild-resistant plastic screw cap. The label applied to the containerincluded the drug name, dosage strength, lot number, storageinstructions, amount of suspension per container, and the manufacturer'sname.

E) Gelatin Capsule (300 mg CPX unit dose)

F) Water Formulation

2. EXAMPLE 2 CPX Absorption Profile in Rats Comparing Water and Corn OilDrug Formulations

This EXAMPLE describes the pharmacokinetics of CPX absorption in ratsfollowing oral administration comparing two different liquidformulations containing the CPX drug, and demonstrates one of theadvantageous properties of a corn oil CPX formulation over a water(i.e., aqueous) CPX formulation.

Experimental—This study was designed to determine the pharmacokineticsof CPX bioavailability in blood plasma following a single oraladministration by oral gavage to male Sprague-Dawley rats. The CPXcompound was suspended in either a corn oil or water formulation andadministered once via oral gavage at 10 ml/kg of rat weight to twogroups of male Sprague-Dawley rats. The two experimental groups aredescribed in TABLE 1 below. TABLE 1 CPX Dosage Number of Animal Group(mg/kg) Vehicle Individuals Identification Nos. 1 1.4 corn oil 1017869-17878 2 1.4 water 10 17879-17888

There was no mortality or signs of morbidity noted at any time duringthe course of this experiment. Blood was collected from the first fiveanimals in each group at 0.25, 1, 4 and 12 hours following dosing, andfrom the second five animals in each group at 0.5, 2, 8 and 24 hoursfollowing dosing. The rats were not fasted prior to blood collection.Approximately 0.4 ml of whole blood was collected from each animal intoheparinized tubes via puncture of the orbital sinus under 70% CO₂/30% O₂anesthesia. Approximately 0.2 ml of plasma was separated bycentrifugation and analyzed for CPX concentration using high performanceliquid chromatography (HPLC) with mass spectrometric detection.Following the completion of blood collection, all surviving animals wereeuthanized by carbon dioxide overdose.

Results/Conclusions—The results of this plasma CPX concentrationanalysis are shown in TABLE 2 below. In TABLE 2, CPX quantitation isshown in ng/ml. The ten individuals receiving the corn oil CPXformulation are shown in the top rows, while the ten individualsreceiving the water CPX formulation are shown in the bottom rows. TABLE2 Animal ID Time in hours No. 0.25 0.5 1 2 4 8 12 24 17869 18.46 — 23.78— 21.17 — BLQ — 17870 BLQ — BLQ — 15.05 — BLQ — 17871 12.33 — 18.07 —27.87 — BLQ — 17872 13.67 — 19.30 — 14.92 — BLQ — 17873 BLQ — BLQ — BLQ— BLQ — 17874 — 13.08 — 18.02 — BLQ — BLQ 17875 — 10.31 — 12.52 — BLQ —BLQ 17876 — 11.91 — 17.60 — 12.49 — BLQ 17877 — 12.85 — 11.09 — 10.31 —BLQ 17878 — BLQ — 13.34 — 12.62 — BLQ 17879 BLQ — BLQ — BLQ — BLQ —17880 BLQ — BLQ — BLQ — BLQ — 17881 BLQ — BLQ — BLQ — BLQ — 17882 BLQ —BLQ — BLQ — BLQ — 17883 BLQ — BLQ — BLQ — BLQ — 17884 — BLQ — BLQ — BLQ— BLQ 17885 — BLQ — BLQ — BLQ — BLQ 17886 — BLQ — BLQ — BLQ — BLQ 17887— BLQ — BLQ — BLQ — BLQ 17888 — BLQ — BLQ — BLQ — BLQQuantitation in ng/ml.BLQ = Below Limit of Quantitation— = No Sample Expected

Rat plasma CPX concentration as a function of time (in hours) using thetwo formulations was measured. Each rat in this experiment (n=10)received a 1.4 mg/kg CPX dose, which was equivalent to a 100 mg dose.Ten rats were used at each time point to generate a mean CPXconcentration value. The results of this experiment are depictedgraphically in FIG. 1.

As can be seen in TABLE 2 above and in FIG. 1, the differences insystemic CPX concentrations between the water and corn oil formulationsis striking. The corn oil group displayed between 10 and 27 ng/ml plasmaCPX as long as 8 hours following drug delivery, while no individuals inthe water vehicle group displayed any detectable plasma CPX. Thus, thecorn oil vehicle formulation provided great benefit over the aqueousvehicle formulation as measured by CPX bioavailability.

3. EXAMPLE 3 CPX Absorption Profiles Comparing Various CPX DrugFormulations in Dogs

This EXAMPLE describes the pharmacokinetics of CPX absorption in dogs asmeasured in blood plasma following oral administration comparing fourdifferent CPX liquid formulations, and demonstrates one of theadvantageous properties of a corn oil CPX formulation over other CPXformulations.

Experimental—This study was designed to determine the relativebioavailability of a single oral CPX dose when administered by gavage tomale Beagle dogs. The CPX dosages was administered in a single 30 mg/kgoral dose in four different suspension formulations. These suspensionformulations were:

-   -   1) xanthan gum (homogenized),    -   2) sodium carboxymethylcellulose [NaCMC] (homogenized),    -   3) sodium carboxymethylcellulose [NaCMC] (non-homogenized), and    -   4) corn oil (homogenized).

Each formulation contained CPX at a nominal concentration of 60 mg/gramof suspension. Nominal doses of 30 mg/kg animal weight were administeredfor each formulation, and were administered gravimetrically at 0.5 g/kg(approximately 6.0 g/dog) by gavage. A total of four male beagle dogswere used in the study. The analysis of each formulation comprised datafrom four dogs (n=4). A combination of naive and non-naive dogs wereused, and a one week “washout period” was maintained between eachformulation trial. Animals were fasted overnight prior to each doseadministration. In one experiment using the NaCMC homogenizedformulation, animals were inadvertently not fasted (data indicatedbelow).

Whole blood samples were collected (approx. 3 ml/sample) by jugularvenipuncture into sodium heparin-containing collection tubes. Sampleswere collected at times 0 (predose) and at 0.25, 0.5, 1, 2, 3, 4, 6, 8,12 and 24 hours after dosing. The whole blood was centrifuged to isolateplasma, and concentrations of CPX in the plasma at these time intervalswere determined using high performance liquid chromatography (HPLC) withmass spectrometric detection, with a lower quantitation limit of 1ng/ml.

The following pharmacokinetic parameters were determined for eachexperimental CPX formulation:

-   -   C_(max)—maximum analyte concentration in the plasma, ng/ml    -   T_(max)—time of maximum analyte concentration in the plasma    -   T_(1/2)—terminal half-life of the drug    -   AUC_(last)—area under the curve (AUC) from time 0 to the last        measurable concentration. The AUC is a measure of total systemic        exposure over a defined time interval. Expressed as ng·h/ml        AUC_((0∞))—area under the curve from time 0 to infinity (also        written AUC_(inf) or AUC_(∞))

Results/Conclusions—No adverse effects were apparent after oraladministration of any of the CPX formulations. The CPX concentrationvalues (ng/ml) at each time point were determined for each dog in thisstudy are shown in FIG. 2. Time is shown in hours, and each of the fourdogs is indicated by its Identification Number. It was observed thatmeasured peak concentrations of CPX in the plasma occurred within 5hours of oral administration dosing.

The data in FIG. 2 was condensed by determining the mean CPX plasmaconcentration (ng/ml) for the group of dogs receiving the same drugformulation (n=4). This mean was calculated for each time point. Themean CPX concentration values are summarized in TABLE 3, below. Alsoincluded are a single set of data for dogs that received the homogenizedNaCMC formulation that were inadvertently not-fasted (i.e., the dogswere fed). TABLE 3 Plasma CPX Concentration (ng/ml ± SEM^(a)) NaCMCNaCMC time xanthan gum NaCMC (non- corn oil (homogenized, (hours)(homogenized) (homogenized) homogenized) (homogenized) fed) 0 BLQ^(b)BLQ BLQ BLQ BLQ 0.25  13.70 ± 11.53 8.48 ± 5.61 3.60 ± 2.16  5.28 ± 3.4818.95 ± 12.67 0.5  25.21 ± 20.09 11.95 ± 9.37  6.08 ± 5.29  47.39 ±87.14 44.79 ± 42.68 1 12.81 ± 6.28 11.20 ± 8.92  1.68 ± 1.46  317.47 ±426.08 52.96 ± 59.44 2  8.04 ± 4.64 5.82 ± 4.53 1.14 ± 0.81  229.23 ±186.06 49.67 ± 63.20 3  4.82 ± 2.35 2.36 ± 1.39 BLQ  79.65 ± 55.05 40.55± 53.08 4  3.53 ± 1.16 1.69 ± 1.44 NC  44.20 ± 31.98  60.62 ± 110.25 6 3.36 ± 3.10 BLQ NC  21.58 ± 13.93 16.24 ± 26.23 8  1.79 ± 1.37 NC^(c)BLQ 10.51 ± 5.98  9.66 ± 16.23 12  28.26 ± 55.28 19.10 ± 35.37 BLQ  5.48± 3.56 3.93 ± 3.82 24  31.29 ± 26.16 43.05 ± 46.86 BLQ  4.60 ± 4.5210.01 ± 10.92^(a)SEM = standard error of the mean^(b)BLQ = Below Limit of Quantitation^(c)NC = mean value not calculated (>50% of individual concentrationswere BLQ)

This data in TABLE 3 above is depicted graphically in FIGS. 3 and 4.FIG. 3 plots the mean CPX plasma concentration (ng/ml) of each fasteddog group versus time (in hours), for each formulation, on a linearaxis. Each data point on this graph represents a mean value derived fromfour animals (n=4). Also included are a single set of data for dogs thatreceived the homogenized NaCMC formulation that were inadvertentlynot-fasted (i.e., the dogs were fed). FIG. 4 shows this same data, buton a semilogarithmic concentration scale. As can clearly be seen in bothof these plots, the CPX concentration in the plasma is strikingly higherwhen the corn oil CPX formulation was used, as compared to any of theother formulations.

Pharmacokinetic analysis of this same data was also undertaken. Theresults of this analysis are shown in TABLE 4 below. The standard errorof the mean is also shown. TABLE 4 Formulation NaCMC xanthan gum NaCMC(non- NaCMC corn oil (homogenized, parameter (homogenized) homogenized)(homogenized) (homogenized) non-fasted) C_(max) (ng/ml)^(a) 29.8 ± 17.88.75 ± 5.62 15.4 ± 8.79 408 ± 372 79.0 ± 101  (51.4 ± 44.6) (22.8 ±15.4) (82.8 ± 97.7) t_(max) (hours)^(a) 0.56 ± 0.32 1.38 ± 1.75 0.69 ±0.38 1.25 ± 0.50 1.50 ± 1.68  (9.4 ± 11.1) (12.4 ± 13.4) (7.25 ± 11.3)AUC(0-8 h) 48.7 ± 25.6 13.3 ± 13.3 27.0 ± 20.4 687 ± 578 284 ± 406 (ng ·h/ml) AUC(0-24 h) 398 ± 506 19.2 ± 25.0 276 ± 332 779 ± 615 395 ± 449(ng · h/ml)^(a)C_(max) and t_(max) values in parenthesis indicate parameterscalculated with 0-24 hour data (including any elevated terminalconcentration-time points).

From TABLE 4 above, it can be seen that oral administration of the cornoil suspension formulation resulted in systemic CPX exposure which wasat least twofold greater than any other formulation tested. Based onplasma AUC(0-8) and C_(max) comparisons of the formulations tested, theoral bioavailability was highest with the corn oil formulation followedin decreasing order by xanthan gum, NaCMC (homogenized), and lastly,NaCMC (non-homogenized).

CPX systemic exposure following administration of the NaCMC(homogenized) formulation was approximately 20-fold greater innon-fasted dogs compared to fasted dogs.

In dogs, C_(max) was 0.4 μg/ml following a 30 mg/kg dose of CPX in thecorn oil formulation. This is in contrast to 0.1 μg/ml observed inprevious dog studies using a methyl cellulose formulation.

Thus, the use of a corn oil CPX delivery formulation results in greatermaximal drug concentration and greater overall systemic drug exposurecompared to any other formulation tested.

5. EXAMPLE 4 CPX Absorption Profile in Humans Comparing Gelatin Capsuleand Corn Oil Formulations

This EXAMPLE describes the pharmacokinetics of CPX absorption in humansfollowing oral administration of two different drug formulations,namely, a gelatin capsule formulation and a corn oil formulation, anddemonstrates the advantageous properties of a corn oil CPX formulationover a standard gelatin capsule formulation.

Expermental—CPX was supplied in two different formulations. These were asuspension in corn oil containing 60 mg of CPX per gram of thesuspension, as described in EXAMPLE 1, and a hard gelatin capsule.

A single 300 mg oral dose of CPX was administered to the subjects inthis experiment. The 300 mg CPX dose was contained in either the cornoil formulation (i.e., 5 ml dosages of 60 mg/ml formulation) or a hardgelatin capsule formulation. The gelatin capsule formulation wasadministered to cystic fibrosis patient subjects (n=4), and the corn oilformulation was delivered to normal male subjects (n=3).

Blood samples were collected for determination of plasma CPXconcentration. For each group, 10 ml samples were collected byindwelling catheter or by venipuncture from the appropriate vein intosodium heparin collection tubes. For each individual, whole bloodsamples were collected predose (t-0), 20 minutes, 40 minutes, and 1,1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 32 and 48 hours followingadministration. The blood samples were centrifuged to isolate plasma,and concentrations of CPX in the plasma at these time intervals weredetermined using high performance liquid chromatography (HPLC) with massspectrometric detection, with a lower quantitation limit of 1 ng/ml.Using these CPX concentration values, pharmacokinetic analysis wasconducted.

Results/Conclusions—No adverse effects were reported after oraladministration of either CPX formulation. A graphical representation ofthe plasma CPX concentrations that were measured in this experiment areprovided in FIG. 5. As can be seen in this FIG., the CPX concentrationsin the subjects receiving the corn oil formulation reach a statisticallysignificant higher level, and reach a C_(max) value much quickercompared to the concentration values in the subjects receiving thegelatin capsule CPX formulation.

Results of the pharmacokinetic analysis are shown in TABLE 5, below.Standard deviation values of the means are also indicated. TABLE 5Single 300 mg CPX Dose Corn Oil Formulation CF patients (n = 4) NormalMales (n = 3) C_(max) (mean), ng/ml 259 ± 191 676 ± 154 C_(max) range144-543 544-845 AUC_(inf) (mean), 1217 ± 824  2621 ± 506  ng · h/mlAUC_(inf) range  581-2423 2061-3043 T_(1/2) (mean), hours 8.5 ± 4.8 13.7± 5.3 

Thus, as can be seen in TABLE 5, the corn oil formulation of CPXprovided at least a two-fold greater maximal plasma CPX concentration,at least double total systemic CPX exposure (as measured by AUC_(inf)),and a longer half-life of the drug in the blood plasma (as measured byT_(1/2)).

7. EXAMPLE 5

(a) CPX Phamacokinetic Clinical Studies in Humans Using a Corn Oil DrugFormulation

This EXAMPLE provides a protocol for the further analysis of safety andpharmacokinetic behavior of CPX when administered to humans in a cornoil vehicle for oral administration. The corn oil suspension used inthis study is the same as described in EXAMPLE 1. The goals of thisprotocol are

-   -   (1) to define the CPX corn oil oral suspension dose which        achieves a maximal AUC of approximately 3275 ng·h/ml, and        simultaneously is safe and tolerable,    -   (2) to characterize the safety and tolerance of CPX corn oil        formulations required to achieve an AUC up to approximately 4500        ng·-h/ml,    -   (3) to compare the concentration versus time profiles of plasma        CPX concentration following administration of a corn oil-CPX        formulation following a high-fat breakfast compared to        administration under fasted conditions, and    -   (4) to define safe and tolerable corn oil-CPX dosage regimens        that result in steady-state trough CPX levels that exceed 300        ng/ml.

Parts (1), (2) and (3) of this study are conducted as a single-blind,randomized, placebo controlled single dose, pharmacokinetically guideddose escalation study. Administration of the corn oil formulations(CPX-containing or placebo) is directly into the subjects mouth vial anoral syringe, followed by the ingestion of 240 ml of water. Differentgroups of four subjects are to receive either a placebo (n=1) or one ofup to six doses of CPX corn oil suspension (n=3) under fasted conditionstargeted to achieve a maximum AUC up to approximately 4500 ng·hr/ml.Upon completion of the highest dose group, a different group of foursubjects repeats one of the doses given previously, in order to increasethe number of subjects for analysis. In addition, a different group offour subjects is administered either placebo (n=1) or one of the dosesof CPX oral suspension (n=3) given previously with a high-fat breakfast.Up to eight groups of four subjects participate in this phase of thestudy.

The first dose used in the study is 30 mg (0.5 grams of an oralsuspension containing 60 mg of CPX per gram of the suspension). Providedthat no dose-limiting adverse effects are observed, dose escalation ispharmacokinetically guided. If the AUC for the 30 mg dose is less thanor equal to 1000 ng·hr/ml (approximately one-third of the maximum AUCobserved as safe and tolerable in the previous Phase I single dosestudy), the second dose is 100 mg. Otherwise, the second dose isselected based on predicted dose to achieve an AUC of approximately 3275ng·hr/mL. If the second dose results in AUC less than 3275 ng·hr/mL, thethird dose is selected to achieve an AUC of approximately 3275 ng·hr/ml.Subsequent dose level(s) to achieve an AUC of up to approximately 4500ng·hr/ml is selected primarily based on pharmacologic effects or adverseevents; however, doses are selected to produce no more than a 33%increase in AUC. Dose groups are evaluated in 7-14 day intervals uponthe condition that the dose given to the previous dose group is deemedsafe and tolerable.

If dose limiting adverse effects are observed in one or more CPX-treatedsubject at a given dose, the next group of four subjects is administeredthe same dose. Should dose-limiting adverse effects not be observed inthe additional dose group, dose escalation resumes. If however,dose-limiting adverse effects are observed in one or more CPX-treatedsubjects in the additional dose group, dose escalation is discontinuedand an optional step-down dose equal to the mid-point between thehighest dose and the previous tolerated dose is given. At any timeduring the study, an individual must be withdrawn from the study in theevent that the subject experiences an intolerable treatment-emergentadverse event as determined by the investigator or subject. Should anintolerable treatment-emergent adverse event or a serious adverse eventattributed to the study drug by the investigator as possible, probableor definite, occur in one or more subject at any time during a dosegroup, the study sponsor and manager jointly determine whether todiscontinue the dose group. The dose escalation is adjusted from theoriginal plan upon discussion with the study sponsor.

Part 4 of the study (i.e., determination of safe and tolerable cornoil-CPX dosage regimens that result in steady-state trough CPX levelsthat exceed 300 ng/ml) is conducted as a single-blind, randomized,placebo-controlled, multiple dose study. Different groups of eightsubjects receive either placebo (n=2) or one of two dosage regimens ofCPX oral corn oil suspension (n=6). The first dosage regimen is selectedto achieve steady state trough plasma CPX concentrations of 300 ng/ml,assuming that the predicted AUC does not exceed values deemed safe andtolerable in the first phase of the study. The second dosage regimen isselected to achieve steady trough plasma CPX concentrations of 600ng/ml, assuming that the predicted AUC does not exceed values deemedsafe and tolerable in the first phase of the study. A new dosage regimenwill not be evaluated until the previous dosage regimen is deemed safeand tolerable.

At any time during the study, an individual must be withdrawn from thestudy in the event that the subject experiences an intolerable treatmentemergent adverse event as determined by the investigator or subject.Should dose limiting adverse effects occur in two or more CPX-treatedsubjects on a given dosage regimen, or an intolerable treatment-emergentadverse effect or a serious adverse effect attributed to the study drugby the investigator as possible, probable or definite, occur in one ormore subject at any time during a dose group, the study sponsor andmanager will jointly determine whether to discontinue the dose group.The selection of dosage regimen may be adjusted from the original planupon discussion with the study sponsor.

For the pharmacokinetic analysis in parts (1), (2) and (3), bloodsamples will be collected prior to dose and 20 and 40 minutes and 1,1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 32 and 48 hours followingadministration of the single oral dose. CPX concentration in the bloodplasma will be determined for each sample. For the pharmacokineticanalysis in part (4), blood samples will be collected prior to the firstand last dose and 20 and 40 minutes and 1, 1.5, 2, 3, 4, 6, 8, 10, 12,16, 24, 32 and 48 hours following the last dose for determination CPXconcentration in the blood plasma. Predose samples will be collectedprior to the first dose given on days 4, 5 and 6. Additional samples arecollected after the first dose given on day 4 at 2, 4, 6, 10 and 12hours post-dose. Pharmacokinetic data for each CPX dose will besummarized using descriptive statistics.

All of the references identified herein, including patents, patentapplications, and publications, are hereby incorporated by reference intheir entireties.

While the invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred method, compound, and composition canbe used and that it is intended that the invention can be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications encompassed within the spirit andscope of the invention as defined by the following claims.

8. A liquid pharmaceutical formulation suitable for oral administrationcomprising an effective amount of a therapeutically active xanthinederivative, substituted xanthine, or pharmaceutically acceptable salt ofsaid xanthine derivative or said substituted xanthine, having theformula:

(I), wherein R₁ and R₂ are the same or different and are C(1-6)alkyl orC(1-6)alkenyl, or hydrogen; R₃ is C(1-6)alkyl or hydrogen, and R₄ isC(48)cycloalkyl, aryl or hydrogen, wherein at least one of R₁, R₂ and isother than hydrogen, in admixture with a pharmaceutically acceptableoil.
 9. The formulation of claim 1, wherein said formulation is asolution.
 10. The formulation of claim 1, wherein said xanthinederivative, substituted xanthine, or pharmaceutically acceptable salt ofsaid xanthine derivative or said substituted xanthine, is hydrophobic.11. The formulation of claim 1, wherein said formulation is asuspension.
 12. The formulation of claim 4, wherein said suspension issubstantially homogenous.
 13. The formulation of claim 4, wherein saidtherapeutically active xanthine derivative, substituted xanthine, orpharmaceutically acceptable salt of said xanthine derivative or saidsubstituted xanthine, is in the form of particles having a mean diameterless than about 100 microns.
 14. The formulation of claim 1 furthercomprising a pharmaceutically acceptable preservative.
 15. Theformulation of claim 1 further comprising a pharmaceutically acceptableantioxidant.
 16. The formulation of claim 1 wherein saidpharmaceutically acceptable oil is a vegetable oil.
 17. The formulationof claim 9 wherein said vegetable oil is selected from the groupconsisting of corn oil, almond oil, coconut oil, cottonseed oil, mustardseed oil, olive oil, palm oil, peanut oil, safflower oil, sesame oil,soybean oil, sunflower oil, and partially or fully hydrogenatedderivatives of said oils.
 18. The formulation of claim 10 wherein saidvegetable oil is corn oil.
 19. The formulation of claim 1 wherein insaid formula (I) R₁ and R₂ are the same or different and are C(1-6)alkylor C(1-6)alkenyl; R3 is C(1-6)alkyl or hydrogen, and R4 is C(4-8)cycloalkyl.
 20. The formulation of claim 1 wherein in said formula (I)R₁ and R₂ are the same and are methyl or allyl, R₃ is ethyl,cyclopropylmethyl or hydrogen, and R4 is cyclohexyl, provided that R₁ isallyl when R₃ is hydrogen, and R₁ is methyl when R₃ is ethyl orcyclopropylmethyl.
 21. The formulation of claim 1 wherein in saidformula (I) R₁ and R₂ are both methyl, R₃ is ethyl, cyclopropylmethyl,and R₄ is cyclohexyl.
 22. The formulation of claim 1 wherein in saidformula (I) R₁ and R₂ are allyl, R₃ is hydrogen, and R₄ is cyclohexyl,cyclohexylmethyl, or cycloheptyl.
 23. The formulation of claim 1 whereinin said formula (I) R₁ is methyl, R₂ is allyl, R₃ is cyclopropylmethylor ethyl, and R₄ is cyclohexyl.
 24. The formulation of claim 1 whereinin said formula (I) R₁ and R₂ are the same or different, and are methyl,propyl, allyl or hydrogen; R₃ is methyl or hydrogen, and R₄ iscyclohexyl or cyclopentyl.
 25. The formulation of claim 1 wherein saidsubstituted xanthine of formula (I) is selected from the groupconsisting of 1,3-dipropyl-cyclopentylxanthine (CPX),1,3-diallyl-cyclohexylxanthine (DAX/DCHX),1,3-dipropyl-7-methylcyclopenthylxanthine (DP-CPX), cyclohexylcaffeine(CHC), and xanthine amino congener (XAC).
 26. The formulation of claim18 wherein said substituted xanthine of formula (I) is1,3-dipropyl-8-cyclopentylxanthine (CPX).
 27. A method for theactivation of ion efflux in ion efflux deficient cells, comprisingcontacting said cells with an effective amount of a liquidpharmaceutical formulation according to one of claims 1 through
 19. 28.The method of claim 20 wherein said cells are cystic fibrosis (CF)cells.
 29. The method of claim 21 wherein said cells have the CFTR-ΔF508mutation.
 30. A method for the treatment of a chronic obstructive airwaydisorder, comprising administering to a subject in need atherapeutically effective amount of a liquid pharmaceutical formulationaccording to one of claims 1 through
 19. 31. The method of claim 23wherein said chronic obstructive airway disorder is characterized bydefective ion transport associated with reduced or abnormal CFTRactivity.
 32. The method of claim 24 wherein said disease or conditionis cystic fibrosis.
 33. An article of manufacture comprising: a) acontainer; b) a liquid pharmaceutical formulation according to one ofclaims 1 through 19 within said container; and c) directions foradministration of said formulation for the treatment of a disease orcondition characterized by defective ion transport associated withreduced or abnormal CTFR activity.